Relationship among LRP1 expression,Pyk2 phosphorylation and MMP‐9 activation in left ventricular remodelling after myocardial infarction

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low‐density lipoprotein receptor‐related protein 1 (LRP1) and MMP‐9 and MMP‐2 spatiotemporal expression after MI. Real‐time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri‐infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri‐infarct and infarct areas. LRP1 also colocalized with proline‐rich tyrosine kinase 2 (pPyk2) and MMP‐9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP‐9 activity in fibroblasts, without significant changes in MMP‐2 activity. MMP‐9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP‐9 up‐regulation associated with ventricular remodelling after MI.     

Prostacyclin production by rat aorta "in vitro" is increased by the combined action of dipyridamole plus pentoxifylline

The present study evaluates the effect of dipyridamole and pentoxifylline, individually and in combination, on PGI2-like production and arachidonic acid metabolism of rat aorta "in vitro". Pentoxifylline 100 microM and dipyridamole 92 and 184 microM increased PGI2-like activity, as measured by the platelet aggregation inhibitory capacity of the aortic ring incubates, by 71%, 46% and 60% respectively; a greater increase in PGI2-like activity was observed with the combination of the drugs than when they were used separately. This effect was observed even at the lowest doses assayed. In fact, dipyridamole 9.2 microM plus pentoxifylline 1 microM increased the PGI2-like activity by 30% while the individual increase was 4.5% and 10.6% respectively. To obtain more information on the effect of the dipyridamole-pentoxifylline combination on arachidonic acid metabolism, arteries were incubated with (1-14C) arachidonic acid, and the 6-keto-PGF1 alpha and PGE2 quantified. Dipyridamole 92 microM plus pentoxifylline 1 and 10 microM increased 6-keto-PGF1 alpha and PGE2 production by about 30% and 48% respectively while the combination with pentoxifylline 100 microM increased the 6-keto-PGF1 alpha 76.5% and the PGE2 50%. The possible biological effect and therapeutic implications of increased PGI2 production by the arteries due to the dipyridamole-pentoxifylline combination remains to be ascertained.     

A single-chain tetradomain glycoprotein hormone analog elicits multiple hormone activities in vivo

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific beta subunit genes to the common alpha subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) beta, follicle-stimulating hormone (FSH) beta, and chorionic gonadotropin (CG) beta subunits to the alpha subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHbeta knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.     

Ganglioside and phospholipid composition of forebrain,cerebellum, and brain stem from adult and newborn rats

The aim of this study was to elucidate whether sex or pregnancy state might affect the content and/or pattern of gangliosides from the forebrain, cerebellum and brain stem of rats. Adult male, mother (1-day after offspring) and nonpregnant rats of similar age were analyzed. Non-significant differences in ganglioside concentrations and patterns were found for the respective neural area of adult male and female rats except for a decrease in cerebellum and brain stem content from mothers and 12.0 months-old males, respectively. Thus, it seems that neither sex nor pregnancy hormones affect these parameters. By contrast, significant differences were found for pattern and ganglioside contents between adult (male and female) rats and newborns (1 day-old). Newborns showed a significant decrease in their forebrain (2.5-fold), cerebellum (2.0-fold) and brain stem (2.0-fold) ganglioside content when compared with adult (male and female) rats. Significant increases (p<0.001) were found in the phospholipid and cholesterol contents in the different brain areas in mothers versus their newborns. The phospholipid pattern also showed significant changes in all brain areas, with an increase (p<0.001) in phosphatidylethanolamine percentage in adult animals, among the main variations. An explanation for these facts is suggested.Abbreviations NeuAc N-acetylneuraminic acid - TLC thin layer chromatography - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - PI phosphatidylinositol - PA phosphatidic acid - SM sphingomyelin - PG phosphatidylglycerol Special issue dedicated to Dr. Santiago Grisolía.     

Metal Uptake and Distribution in Cultured Seedlings of Nerium oleander L. (Apocynaceae) from the Río Tinto (Huelva, Spain)

Nerium oleander L. (Apocynaceae) is a micro-nano phanerophyte that grows in the riverbanks of the Río Tinto basin (Southwest Iberian Peninsula). The waters and soils of the Río Tinto area are highly acidic and have high concentrations of heavy metals. In this environment, N. oleander naturally grows in both extreme acidic (EA) and less extreme acidic (LEA) water courses, excluding, and bioindicating certain metals. In this work, we compared and evaluated the accumulation preferences and capacities, the distribution and processes of biomineralization of metals (Fe, Cu, Zn, Mn, Mg, Ca) in the first stages of growth of EA and LEA oleanders by means of inductively coupled plasma–mass spectrometry, scanning electron microscopy, and energy dispersive X-ray analyzer analysis. Seeds from both environments were grown and treated with a self-made solution simulating the most extreme red waters from the Río Tinto. LEA plants drastically reduces the metal uptake at the beginning, but later reactivates the uptake reaching concentration values in the same range as the EA plants. The results showed high Mn, Zn and Mg concentrations, accumulation of Fe and Cu in plants from both environments, differing from the metal concentrations of field-grown oleanders. Iron bioformations with traces of other metals were present inside and over epidermal cells and inside vascular cells of stems and roots. They were absent of leaves. The accumulation properties of N. oleander in its early stages of development make it a species to take in consideration in phytoremediation processes but optimized conditions are needed to ensure enough biomass production.     

An Improved Semiquantitative Method for Elemental Analysis of Plants Using Inductive Coupled Plasma Mass Spectrometry

A semiquantitative ICP-MS method suitable for evaluating metal content in plants exposed to high metal concentrations is described. The methodology which has been tested using different plant reference material is able, in only a few minutes, to obtain qualitative and quantitative information from the sample. Recoveries close to 100% were obtained for more than half of the referenced elements with an RSD <10%, thus avoiding the generation of a calibration line for each of the elements to be analyzed. The use of multi-standards improves the results obtained with conventional standards by a factor of two. In these conditions the semiquantitative multi-elemental analysis is a valid alternative for most of the analyzed elements with the exception of Si, Al, and P which requires the use of a quantitative ICP-MS analysis for its determination. Two different digestion protocols were tested, with and without the addition of HF to the HNO(3)?+?H(2)O(2) extraction mixture. Similar results were obtained for poplar and bush reference materials using the HNO(3)?+?H(2)O(2) mixture. The addition of HF did not improve the recovery of different elements, in general, but is critical for the quantitative measurements of Al and Si as its presence allows results to be obtained with an RDS <5%.     

Selection of putative colorectal cancer markers by applying PCA on the soluble proteome of tumors: NDK A as a promising candidate

Aiming to find new tumor markers for colorectal cancer (CRC), we applied proteomic methodologies to compare the soluble sub-proteome of healthy and tumoral colorectal mucosa. Out of 91 differentially expressed proteins, 23 were selected by principal component analysis (PCA) as the major contributors to the overall difference detected. After MS/MS analysis, 16 proteins were identified. From those, we chose 14-3-3-zeta/delta, retinoblastoma-binding protein 4 (RBBP-4), DJ-1, and nucleoside diphosphate kinase A (NDK A) for further studies, on the basis of their levels and known implication in cancer. Specific immunodetection demonstrated only the NDK A levels allowed to differentiate healthy mucosa from tumor tissue in all the patients. Hence, we used the colon cancer cell line Caco-2 to study the relationship between NDK A and colon cell tumorigenesis, finding it over-expressed in undifferentiated (tumor-like) cells regarding the differentiated ones. Noticeably, we also found increased levels of the NDK A in the secretome of tumor-like cells and, as expected, indications of higher levels of NDK A in the serum of CRC patients. In conclusion, the four validated proteins could constitute a panel of tissue markers for CRC, being the NDK A the most interesting candidate for further serum biomarker studies.     

Purification of human alpha-L-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein

Alpha-L-fucosidase (FUC) is a glycosidase involved in the degradation of fucose-containing glycoconjugates. A cDNA representing the complete sequence of human FUC was inserted into the prokaryotic expression vector pGEX-2T. High levels of the glutathione S-transferase (GST) fusion protein were detected in Escherichia coli cells after induction with isopropyl thio-beta-D-galactopyranoside. The GST-FUC protein was mostly found as inclusion bodies and attempts to optimise its expression as a soluble form were unsuccessful. Nevertheless, the recombinant protein was purified by affinity chromatography on glutathione-sepharose and its fucosidase activity was characterised. After thrombin cleavage of the GST tag, the FUC precursor protein was purified by electro-elution.     

Arachidonate incorporation into rat aorta lipid fractions and eicosanoid formation. Effect of prolonged prostacyclin production

Endogenous arachidonic acid (AA) content, incorporation of radiolabelled AA (AA*) into total lipids, main lipid fractions and different phospholipids (PL), and prostanoid formation have been evaluated in fresh (control) rat arteries and in arteries after 180 min of incubation in buffer (exhausted). The results show that PGI2 formation from endogenous AA decreased 90% in exhausted arteries while AA content decreased only 30%. The total AA* incorporation was significantly higher in exhausted arteries than in controls (p less than 0.01). The distribution of AA* in lipids is altered in exhausted arteries; it increases in total PL, particularly in phosphatidylethanolamine, and decreases in phosphatidylcholine and phosphatidylserine + phosphatidylinositol. AA* content was also lower in triglycerides and esterified cholesterol of exhausted arteries than in control arteries. The AA* metabolized to PGI2 was 83% lower in exhausted arteries than in controls, while PGE2 and TXB2 formation were not modified by the exhaustion process. When the effect that longer incubation in plasma (180 min) has on AA metabolism and turnover was evaluated, PGI2 formation from endogenous AA was found to be increased in comparison with arteries incubated for the de same period in buffer, and the changes observed in the distribution of AA in lipid fractions are smaller than those found in buffer-exhausted aortas. The results of the present study indicate that prolonged production of prostanoids leads to an alteration in AA turnover and to an inactivation of the PGI2-forming system. Plasma seems to protect AA metabolism.